Despite a continuing refinement in our understanding of eukaryotic chromatin, much remains to be learned about chromatin structure and its relationship to gene expression. Histone and HMG (high mobility group) proteins represent the major constituents of the acid extractable chromosomal proteins; additional components have been detected employing high resolution analytical techniques. The structure of these additional basic (or in part basic) chromosomal proteins and their role in chromatin structure and/or function is unknown. This laboratory has isolated to homogeneity one such histone-like protein that is rich in alanine and lysine (protein AK) by high resolution gel filtration of H2SO4 extracts of calf thymus chromatin. A variety of physico-chemical criteria distinguishes it from previously described histones and HMG proteins. The immediate objectives of this proposal are to provide further general characterization of the molecule including the elucidation of significant portions of its primary structure and description of the nature of its interaction with chromatin. The degree of heterogeneity in molecular charge will be estimated by isoelectric focusing. The tissue and species distribution of protein AK will be determined. The relationship of protein AK isolated from calf thymus, protein AK isolated from other tissues and species, and other DNA binding proteins will be examined by amino acid composition analysis, chromatoelectrophoresis of enzymatically derived peptides, and estimation of immunologic cross relatedness. Primary structure analyses initially will involve automated Edma sequencing of the major peptides produced by selective cleavage(s) at tyrosine, methionine and glutamic acid residues. The occurrence of posttranslationally modified residues will be examined as will be the distribution of the protein in nucleosome "core" vs. "linker" regions. Characterization of the interaction of protein AK with chromatin will involve estimation of extractability by salts and hydrophobic bond disrupting solutes and circular dichroism. It is expected that these studies will provide considerable insight into the structure of protein AK and the nature of its interaction with chromatin.